The determination of the hematocrit of blood is clinically important for the detection of hemorrhage and anemia. This measurement is usually made by centrifuging a blood sample and measuring the lengths of the packed red cell column and the total blood column; i.e., red cells and plasma. The hematocrit is then calculated as the ratio of the red cell column length to the length of the total blood column. The centrifugation method provides a reasonably accurate measure of the hematocrit, but is tedious and time consuming; e.g., the so-called microhematocrit method, in which a capillary tube is filled with blood by capillary action and sealed at one end, requires centrifugation at 10,000 to 12,000 g for 5 to 10 minutes.
Another method for the determination of hematocrit utilizes a flow-through cell analyzer; i.e., a flow cytometer. This instrument has been described in various patents, including U.S. Pat. Nos. 3,275,834 to Stevens and 2,565,508 to Coulter. The actual detection in these systems can be an electrical impedance measurement or an optical measurement.
The principle utilized in electrical impedance flow cytometers; e.g., Coulter Counter, involves passing a diluted blood sample through an aperture through which a current is flowing between two electrodes. The passage of a single cell through the aperture results in a voltage pulse which is proportional in magnitude to the volume of the cell. This approach requires a precise, large dilution (1:50,000) of the blood sample and is subject to inherent errors depending on the shape and electrical resistance of the cell and coincidence loss.
In optical-based flow cytometers, as described in the Stevens patent, a diluted blood sample passes through a sensing zone which is defined by a light beam. The optical measurement detects light scattering either from external reflections from the surface of the cell, from transmitted and refracted light passing through the cells, or from diffracted light which has passed tangential to the cell surfaces. This approach suffers from the same limitations as the impedance method in that the optical signals depend on the cell shape and refractive index, as well as the cell volume. Coincidence errors can also occur. Attempts to improve these methods by diminishing these errors have been reported; e.g., W. P. Hansen, Canadian Pat. No. 1144-280.
Another method for the measurement of hematocrit is based on a conductance measurement of the blood sample as described in A. Slawinski, Biochem. J. 27, 356 (1933); J. A. Kernen et al. J. Lab. Clin. Med. 57, 635 (1961); R. H. Okada et al. IRE Transactions on Medical Electronics 1960, 188; R. E. Davis, Lab. Practice 15, 1376 (1966); and H. Kiesewetter, German Patent No. 3202-067-A. This method is based on the principle that the conductance of a whole blood sample is inversely proportional to the cell volume. This method is subject to serious errors due to temperature changes, nonlinear calibration, and abnormally high levels of plasma proteins, electrolytes, and white cells in the blood sample.
It is an object of the present invention to provide a simple and rapid electrochemical method to estimate the hematocrit of blood which requires a minimum of sample manipulation and is not subject to errors caused by differences in cell shape and abnormal levels of electrolytes and proteins in the blood sample.